Dynamic characterization of outer-membrane-protein biogenesis by the BAM and TAM complexes

项目摘要/摘要 传染病是世界上第二大死亡原因。新的抗生素类药物 几乎不存在，病原菌对现有细菌的抗药性迅速增加，发展成- 开发新的方法正成为推进人类健康努力的当务之急。分子建模 将在这些新方法中发挥至关重要的作用，因为关键的 细菌蛋白质和抗菌剂的结构、过程和相互作用。 为了说明这些结构和过程，PI将重点介绍革兰氏阴性的Definning特征 细菌，即它们的第二外膜，以及完整的膜蛋白是如何插入到那里的。这些 外膜蛋白(Omp)，几乎所有的都属于一种特殊的fic类，称为-桶，利用 插入的两个关键系统：生存必不可少的BAM系统和相关的系统，必要的 因为他有毒力。利用这些系统作为抗菌目标需要全面了解 结构、动力和功能之间的关系。在fi第一个目标中，一种新的机制，其中的密钥 每个系统的组件BAMA和TAMA分别通过其自身的增强来催化插入 -将对桶进行评估。在分子动力学模拟中将产生插入的中间态 并通过二次fi去交联法和电生理学方法进行了实验测定。在 第二个目标，巴马和多玛如何扰乱膜，本身是插入过程的积极参与者，将 要下定决心。模拟表明存在膜缺陷，这种缺陷是由于异常的 巴马-桶的薄而不稳定的部分；突变以改变这种扰动，并可能减少 fi插入效率将在电子计算机中预测，并在体内进行测试。最后，在第三个目标中， 将描述BAMA和TAMA以及BAMA与其他BAM组件的交互作用。基于 根据观察到的构象变化，将选择限制构象的小分子候选药物 fl的灵活性和/或抑制BAMA或TAMA与其他复杂成员的结合。然后，这些候选人将接受测试 用于抗菌活性的实验。

Project Summary/Abstract Infectious diseases are the second leading cause of death in the world. With novel classes of antibiotic drugs virtually nonexistent, and the resistance of pathogenic bacteria to current ones increasing rapidly, the develop- ment of new approaches is becoming an imperative for advancing human health efforts. Molecular modeling will play an essential role in these new approaches, due to the fundamentally atomic-scale nature of the critical structures, processes, and interactions underlying the function of both bacterial proteins and antibacterial agents. In order to illuminate these structures and processes, the PI will focus on a defining feature of Gram-negative bacteria, namely their second, outer membrane, and how integral membrane proteins are inserted there. These outer-membrane proteins (OMPs), practically all of which belong to a specific class known as -barrels, utilize two key systems for insertion: the BAM system, essential for viability, and the related TAM system, necessary for virulence. Exploiting these systems as antibacterial targets requires a comprehensive understanding of the relationship between structure, dynamics, and function. In the first aim, a novel mechanism in which the key component of each system, BamA and TamA, respectively, catalyzes insertion through augmentation of its own -barrel will be evaluated. Intermediate states of insertion will be generated in molecular dynamics simulations and assayed experimentally through both disulfide cross-linking and electrophysiology measurements. In the second aim, how BamA and TamA perturb the membrane, itself an active participant in the insertion process, will be determined. Simulations have indicated the existence of a membrane defect that forms due to an unusually thin and unstable part of the -barrel of BamA; mutations to alter this perturbation, and presumably decrease OMP insertion efficiency, will be predicted in silico and tested in vivo. Finally, in the third aim, the dynamics of BamA and TamA as well as BamA's interactions with other BAM components will be characterized. Based on the conformational changes observed, small-molecule drug candidates will be selected that limit conformational flexibility and/or inhibit binding of BamA or TamA to other complex members. These candidates will then be tested experimentally for antibacterial activity.